Studies from this laboratory and others have documented an association between cellular Ca2+ loading and cardiac myocyte death following exposure to anoxia. It remains unclear whether these changes in Ca2+ mediate cell injury and how this may occur. We characterized the changes in Ca2+ which occurred in the cytosolic compartment [Ca2+]c and mitochondrial compartment [Ca2+]m of adult rat cardiac myocytes exposed to hypoxia/reoxygenation to define the relation between subcellular Ca2+ regulation and cell injury. During anoxia, [Ca2+]c and [Ca2+]m rose only after the onset of ATP-depletion rigor contracture. At reoxygenation 35 minutes later, [Ca2+]c fell rapidly to preanoxic levels while [Ca2+]m fell more slowly. Cellular hypercontracture or death was associated with a rise in [Ca2+]c or [Ca2+]m to greater than 250 nM just prior to reoxygenation. Reversible relatively minor rises in [Ca2+]m may be seen and do not predict cell death. The cause of cell death remains unclear but does not appear to be the result of massive reoxygenation-induced Ca2+ overload.